Red blood cell trapping in the kidney vasculature promotes mitochondrial dysfunction independent of ischemia time; evidence for heme toxicity

Acute kidney injury (AKI) is a major clinical problem that is estimated to kill 2 million people per year. Red blood cell (RBC) trapping is a hallmark finding in ischemic AKI in humans and animal models. RBC trapping presents as accumulation of of RBCs in the outer-medullary vasculature following a period of ischemia. Our group has recently shown that RBC trapping promotes tubular injury independent of ischemia time in rats, however, the mechanism through which this occurs remains unknown. We hypothesize that RBC trapping directly promotes tubular injury through the breakdown of trapped RBCs which results in the extravasation of toxic heme, which is then taken up by the tubular mitochondria causing cellular injury. Our study was performed in age matched male and female Sprague Dawley rats 10-12 weeks of age. Rats were anesthetized with isoflurane (2-5%) and a midline incision performed. The left and right renal arteries and veins were then isolated. To promote ischemia with RBC trapping a vessel clamp was placed on the renal vein. To promote ischemia without RBC trapping the clamp was placed on the artery. Body temperature was maintained at ~37C using a heating pad and lamp and clamps were left in place for 45 minutes before harvesting the kidneys without reperfusion. Mitochondria were then isolated from the kidney cortex and medulla and mitochondrial membrane potential (TMRM) and hydrogen peroxide production (Amplex Red) were then determined. Clamping of the renal artery (n=7) or vein (n=9) or control (unclamped, n=7) was alternated between left and right kidneys and between animals. In the medulla of female rats, renal arterial occlusion without reperfusion resulted in a significant decrease in mitochondrial membrane potential when compared to controls (p<0.001) which as reversed with venous clamping (p=0.001). Similar responses were observed in male rats. Compared to controls, in the outer-medulla of female rats, renal arterial occlusion resulted in a significant increase in hydrogen peroxide production (p<0.001). In contrast, in kidneys with venous clamping, hydrogen peroxide production was not increased (females) or significantly decreased (males, p<0.0001). Our data indicate that mitochondrial membrane potential and hydrogen peroxide production differ dependent on whether ischemia was caused by arterial (ischemia without RBC trapping) or venous (ischemia with RBC trapping) clamping. This indicates that alternative mechanisms mediated mitochondrial dysfunction when RBC trapping is present. Increased mitochondrial membrane potential is consistent with mitochondrial iron overload from heme uptake. While increases in mitochondrial membrane potential are normally associated with increased reactive oxygen species production, we speculate that innate peroxidase activity of heme is lowering mitochondrial hydrogen peroxide production. Our data are consistent with RBC trapping causing toxic tubular injury following ischemic AKI in rats. AHA Transformative Project Award to Paul O'Connor NIDDK 1R01DK138150-01A1 to Paul O'Connor APS SURF fellowship to Angela Ajith This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.